VHHs, also known as single domain antibodies (sdAbs) or Nanobodies™, are variable domains derived from heavy chain antibodies present in camelids. Recombinant VHHs are obtained by screening libraries prepared from lymphocyte RNA of naïve or immunized animals, they are renowned for their easy cloning and expression, and have many applications in research, therapy and diagnosis (1-3).
Aiming to obtain VHH directed against various antigens present on human red blood cells, the inventors immunized a dromedary by transfusion of human blood and derived a VHH library from the animal's lymphocytes. Since VHH that recognizes indiscriminately red cells of all humans may have interesting applications the inventors screened the library against glycophorin A (GPA) a protein present at a high copy number on red cells and isolated several VHHs: One of them which represented 67% of all isolated sequences was fully characterized and is described.
Glycophorins A (GPA) and B (GPB) are essentially expressed on red cells (even though some expression was detected in renal tissue). Expression level is as high as 800 000 copies per cell for GPA and 200 000 copies for GPB (4-5). The highly homologous GPA and GPB are encoded by two genes derived one from the other after a gene duplication event. The sequence of the 26 N-terminal amino acids of GPB is identical to one of the two alloforms of GPA N-terminus. GPA and GPB are single transmembrane domain proteins with a heavily glycosylated extracellular domain. GPA and GPB do associate in the red cell membrane hence homodimers and heterodimers of GPA and GPB are present in the membrane as shown by SDS polyacrylamide gel electrophoresis of red cells membrane extracts. Glycophorins carry several blood group antigens, the most important of them are M and N blood group antigens on GPA, N, S and s antigens on GPB. Blood group antigens carried by glycophorins are important for transfusion medicine and may be responsible for adverse reactions in case of poorly matched transfusion and also be responsible of hemolytic disease of the newborn. Moreover glycophorins carry antigens which are independent of blood groups and several murine monoclonal antibodies exist that target such epitopes constantly present on the molecules independently of the blood group (6).
The present invention demonstrates that the isolated VHH may be used for autologous red cells agglutination assays (7-9). In short monovalent VHH fused to an antigen added to whole blood taken from a patient may induce red cells agglutination if patient's plasma contains antibodies directed against fused antigen.
Many drugs have limited therapeutic action because they are rapidly eliminated from the body when administered. For example, many polypeptides, peptides or chemical compounds that have therapeutically useful activities are rapidly cleared from the circulation via the kidney. Accordingly, a large dose must be administered in order to achieve a desired therapeutic effect. Accordingly, there is a need of developing fusion proteins or immunoconjugates which because of their properties to interact with red cells may increase drug serum half-life.